To date, studies of human type 1 diabetes (T1D) have failed to provide a mechanistic understanding of the underlying causes of the disease, largely because patients must be analyzed long after initiation of the autoimmune attack. Our ignorance of the key molecules and cells mediating the initiation and progression of human T1D may well underlie the paucity of significant new therapeutic interventions. This renewal proposal addresses the stated goal of RFA-DK-18-013 that ?CMAI supports resource development projects that are primarily focused on the reagents and model systems needed for mechanistic study of human T1D.? Our Scientific Premise is that human T1D beta cells and immune cells transplanted into optimized immunodeficient mice (OPTI-MICE) will provide tractable model systems to study human T1D. We propose short term (Aim 1) and long term (Aim 2) goals. Aim 1 will develop an effector phase model of T1D in OPTI-MICE. Our team has already assembled the 3 key components that are required to develop an effector model of T1D: 1) Appropriate OPTI-MICE as recipients; 2) autoreactive T cells, lines, clones and iPS- derived T cells from T1D donors; 3) autologous human iPS cell-derived (SC)-? cells. We have developed NSG mice deficient in MHC class I and II that do not develop GVHD when engrafted with functional human T cells for this aim. We have also recruited Dr. Sally Kent, an expert in islet autoreactive T cell cloning, to provide T1D autoreactive T cell lines and clones from consented T1D patients. Aim 2 will reconstruct human T1D in OPTI-MICE using cells derived from T1D iPS cells. These iPS cells will be used to produce the three key cell types: hematopoietic stem cells (HSC) that will generate immune systems, thymic epithelial cells (TEC), and ?-cells, all integral to the pathology of T1D. These cells will be derived through the use of directed differentiation and reprogramming strategies. We have been successful in generating functional human ? cells from human control and T1D patient iPS cells, providing a standardized and reproducible source of ? cells for our studies. We have successfully performed directed differentiation of human ES cells to generate HSCs, and will use similar approachs for directed differentiation of iPS cells into HSCs. Functional human TEC will also be generated using directed differentiation protocols similar to those used to achieve fully differentiated human ? cells. Each cell type will be subjected to rigorous analysis in vitro and in vivo to ensure full functionality. Differentiated ?-cells, TECs, and immune cells derived from T1D donors will be co-transplanted into OPTI- MICE specifically optimized to enhance T cell, SC-? cell, SC-HSC, and SC-TEC cell engraftment and function allowing reconstitution of an individual patient's disease in an animal model. These new models of human diabetes will permit detailed observation, manipulation, and analysis of T1D, enabling us to determine the cells and antigens that initiate T1D, drive disease progression and mediate beta cell destruction. We have assembled an collaborative team of scientists that have the expertise required to accomplish this project.